CN117821569A - Biomarker for screening non-starch polysaccharide with hypoglycemic activity and application thereof - Google Patents

Abstract

The invention discloses a set of biomarkers for screening non-starch polysaccharide with hypoglycemic activity and application thereof, belonging to the technical field of food biology. The biomarkers include bacterial biomarkers and metabolic biomarkers; bacterial biomarkers include: faecalis (Faecalibacterium), faecococcus (Coprococcus), bacteroides (bacteriodes), anaerobes (anaerosporides); the metabolite biomarkers include: jiang Tangzhi A, pterin-1-phosphate, kurarinone, tricarballylic acid, N-carbamoylputrescine, N omega-acetylhistamine, tyramine, benzamide. The biological markers can be used for screening the non-starch polysaccharide with high hypoglycemic activity, so that the screening period and the screening cost can be saved, and the rapid research, development, popularization and application of products such as medicines, foods, feeds and the like which take the non-starch polysaccharide as a source are promoted.

Description

A set of biomarkers for screening non-starch polysaccharides with hypoglycemic activity and their applications

Technical Field

The invention belongs to the field of food biotechnology, and particularly relates to a set of biomarkers for screening non-starch polysaccharides with blood sugar-lowering activity and applications thereof.

Background technique

Diabetes is a metabolic disease characterized by high blood sugar levels caused by the body's inability to produce insulin or to fully utilize insulin. It has become one of the most serious global public health threats of our time. According to the International Diabetes Federation, there are 537 million people with diabetes between the ages of 20 and 79, accounting for 10.5% of the global population in this age group. This number is expected to rise to 12.2% by 2045. However, most of the current glucose-lowering drugs used to treat diabetes, such as metformin, have limitations and some side effects. Most diabetes may be caused by unhealthy eating habits. Natural active ingredients in food have few side effects, many effects, and low cost. Taking food active ingredients with blood sugar-lowering properties has become a good means of preventing and treating diabetes.

Polysaccharides are the main active ingredients in edible fungi. Many studies have reported that polysaccharides have activities such as antioxidant, anti-tumor, hypoglycemic, immunomodulatory, and hypolipidemic. They can be used as a dietary supplement to effectively regulate human health. Studies have shown that some non-starch polysaccharides with significant hypoglycemic activity can be used as effective dietary supplements to prevent and improve diabetes, but how to screen and determine the hypoglycemic activity of non-starch polysaccharides remains an unresolved problem. In general, non-starch polysaccharides are not degraded by digestive enzymes in the small intestine. After entering the large intestine, they are fermented and utilized by intestinal microbiota, thereby regulating the composition and structure of intestinal bacteria and the production of metabolites of intestinal flora, and finally regulating host health. The metabolites produced by intestinal flora after utilizing polysaccharides mainly include polyamines, secondary bile acids, short-chain fatty acids, choline metabolites and lipids. These fermentation metabolites have the activity of alleviating and improving diabetes.

At present, the research on the hypoglycemic activity of non-starch polysaccharides mainly relies on animal or cell models, which are relatively time-consuming. The present invention is based on the theory that non-starch polysaccharides regulate the composition of intestinal bacteria and promote them to produce a variety of beneficial metabolites. It aims to link the hypoglycemic effect of non-starch polysaccharides with their intestinal metabolite levels or the abundance of related bacteria, and to find some potential key metabolites and bacteria, which can be used to evaluate the hypoglycemic activity of different edible fungus polysaccharides.

Summary of the invention

The primary purpose of the present invention is to overcome the time-consuming and cumbersome methods of comparing the hypoglycemic activity of non-starch polysaccharides from different sources, determine key biomarkers, and provide a set of biomarkers for screening non-starch polysaccharides with hypoglycemic activity.

Another object of the present invention is to provide the use of the above-mentioned biomarker for screening non-starch polysaccharides with hypoglycemic activity.

The purpose of the present invention is achieved through the following technical solutions:

A set of biomarkers for screening non-starch polysaccharides with hypoglycemic activity, including bacterial biomarkers and metabolic biomarkers; among them,

Bacterial biomarkers include: Faecalibacterium, Coprococcus, Bacteroides, Anaerostipes;

Metabolite biomarkers included: ginger glycolipid A, 1-phosphoterin, matrine, tricarbamic acid, N-carbamylputrescine, Nω-acetylhistamine, tyramine, and benzamide.

The application of the above biomarker for screening non-starch polysaccharides with hypoglycemic activity in screening non-starch polysaccharides with high hypoglycemic activity.

A method for screening non-starch polysaccharides with high hypoglycemic activity comprises the following steps:

(1) In vitro fermentation of non-starch polysaccharides: using the non-starch polysaccharide to be tested as the sole carbon source to prepare a fermentation medium; sterilizing or disinfecting the obtained fermentation medium, inoculating a fecal flora solution and performing in vitro anaerobic fermentation; separating the obtained fermentation liquid into solid and liquid to obtain a solid and a liquid; and setting a control, which is a fermentation medium without non-starch polysaccharides;

(2) Biomarker determination: using real-time fluorescence quantitative PCR to determine the abundance of bacterial biomarkers in the solid obtained in step (1); determining the concentration of metabolite biomarkers in the liquid obtained in step (1); wherein the total abundance of bacterial biomarkers is T1, and the total concentration of ginger lipid A, 1-phosphoterin, matrine, tricarbamic acid, N-carbamyl putrescine, Nω-acetylhistamine, tyramine, and benzamide is T2;

(3) Screening and evaluation: The hypoglycemic activity of the non-starch polysaccharide to be tested is obtained based on the abundance of bacterial biomarkers and the content of metabolite biomarkers combined with the following judgment criteria;

judgement standard:

① If the following conditions are met, the non-starch polysaccharide to be tested is judged to have a strong hypoglycemic effect: T1 ≥ 18.0*TC1 and T2 ≥ 1.18*TC2;

② If the following conditions are met, the hypoglycemic effect of the non-starch polysaccharide to be tested is judged to be moderate: 3.2*TC1≤T1＜18.0*TC1 and 1.14*TC2≤T2＜1.18*TC2;

③ If the following conditions are met, the non-starch polysaccharide to be tested has a weak hypoglycemic effect: 2.40*TC1≤T1＜3.2*TC1 and 1.06*TC2≤T2＜1.14TC2;

④ If the following conditions are met, it is determined that the hypoglycemic effect of the non-starch polysaccharide to be tested is not significant: T1＜2.40*TC1 and T2＜1.06*TC2.

The non-starch polysaccharide described in step (1) is preferably an edible fungus polysaccharide, and the source is preferably at least one of Ganoderma lucidum, Cordyceps militaris, Poria cocos and Lentinula edodes.

When the non-starch polysaccharide described in step (1) is a water-soluble polysaccharide, it is preferably extracted by ethanol defatting, hot water extraction, neutral protease treatment, Sevag method protein removal, ethanol precipitation, dialysis, and freeze-drying; more preferably, it is prepared by the following steps:

1) soaking the edible fungi in an ethanol solution with a concentration of 70 to 90% (v/v) to defatted and dried them to obtain a defatted and dried raw material;

2) mixing the defatted and dried raw material with water in a ratio of 1 g of the defatted and dried raw material to 15 to 30 mL of water, and leaching at 80 to 100° C. to obtain an extract;

3) then adding neutral protease to the extract to carry out enzymatic hydrolysis; reacting at 50°C for 4 hours;

4) removing protein from the enzymatic hydrolyzate obtained in step (3) by the Sevag method, removing organic reagents by a vacuum rotary evaporator and concentrating;

5) precipitating the concentrated solution obtained in step 4) with anhydrous ethanol at 4° C. overnight, collecting the precipitate by centrifugation, and re-dissolving the precipitate by adding water;

6) The supernatant is dialyzed and vacuum freeze-dried to obtain non-starch polysaccharide.

The amount of ethanol solution used in step 1) is based on the ability to immerse the raw material; more preferably, it is calculated based on a material-liquid ratio of 1g:3-5mL; most preferably, it is calculated based on a material-liquid ratio of 1g:4mL.

The concentration of the ethanol solution in step 1) is preferably 80% (v/v).

The soaking time in step 1) is more than 12 hours; more preferably 20 to 28 hours; most preferably 24 hours.

The drying conditions in step 1) are preferably drying at 50-70° C. for 30-90 min; more preferably drying at 60° C. for 60 min.

The ratio described in step 2) is preferably 1 g:20 mL.

The water described in step 2) is preferably deionized water or ultrapure water.

The extraction temperature in step 2) is preferably 100°C.

The extraction time in step 2) is preferably 2 hours.

The number of extractions in step 2) is preferably at least 2 times.

The amount of the neutral protease used in step 3) is preferably 0.3-1% (w/w) based on the concentration of the neutral protease in the extract; more preferably 0.5% (w/w).

The enzymatic hydrolysis reaction in step 3) is preferably carried out at 40-60°C for 3-5 hours; more preferably at 50°C for 4 hours.

The reagent in the Sevag method described in step 4) is preferably a reagent obtained by mixing chloroform and n-butanol in a volume ratio of 4:1.

The number of times of protein removal in the Sevag method described in step 4) is preferably 3 times.

The concentration method in step 4) is preferably rotary evaporation.

The degree of concentration in step 4) is preferably concentrated to 1/3 of the volume of the original solution.

The volume of anhydrous ethanol in step 5) is preferably 3 to 5 times the volume of the concentrated solution; more preferably 4 times the volume of the concentrated solution.

The dialysis bag used in the dialysis in step 6) is preferably a dialysis bag with a molecular weight cutoff of 10,000 Da.

The dialysis time in step 6) is preferably 24 to 72 hours, more preferably 48 hours.

When the non-starch polysaccharide described in step (1) is an alkali-soluble polysaccharide, it is preferably extracted by ethanol defatting, hot water extraction, alkali solution extraction, acid neutralization, dialysis, and freeze-drying; more preferably, it is prepared by the following steps:

A. soaking the edible fungi in an ethanol solution with a concentration of 70 to 90% (v/v) to defatted and dried them to obtain a defatted and dried raw material;

B. Mix the defatted and dried raw material with water in a ratio of 1 g of defatted and dried raw material to 15 to 30 mL of water, and extract at 80 to 100° C. to obtain a precipitate;

C. Leaching the precipitate with an alkaline solution to obtain a supernatant;

D. neutralizing the supernatant obtained in step C with an acid;

E. The neutralized supernatant is dialyzed and vacuum freeze-dried to obtain non-starch polysaccharides.

The amount of ethanol solution used in step A is based on the ability to immerse the raw material; more preferably, it is calculated based on a material-liquid ratio of 1g:3-5mL; most preferably, it is calculated based on a material-liquid ratio of 1g:4mL.

The concentration of the ethanol solution in step A is preferably 80% (v/v).

The soaking time in step A is more than 12 hours, more preferably 20 to 28 hours, and most preferably 24 hours.

The drying conditions in step A are preferably drying at 50-70° C. for 30-90 min; more preferably drying at 60° C. for 60 min.

The ratio described in step B is preferably 1 g:20 mL.

The water described in step B is preferably deionized water or ultrapure water.

The leaching temperature in step B is preferably 100°C.

The extraction time described in step B is preferably 2 hours.

The number of leaching in step B is preferably at least 2 times.

The alkaline solution described in step C is preferably a NaOH solution; more preferably a NaOH solution with a concentration of 0.5 to 1.5 mol/L; most preferably a NaOH solution with a concentration of 1 mol/L.

The amount of the alkaline solution in step C is preferably calculated based on 50-70 mL of alkaline solution per gram of precipitation; more preferably, it is calculated based on 60 mL of alkaline solution per gram of precipitation.

The acid described in step D is preferably hydrochloric acid; more preferably hydrochloric acid with a concentration of 1 mol/L.

The degree of neutralization described in step D is preferably neutralized to neutral.

The dialysis bag used in the dialysis in step E is preferably a dialysis bag with a molecular weight cutoff of 10,000 Da.

The dialysis time in step E is preferably 24 to 72 hours, more preferably 48 hours.

The composition of the fermentation medium described in step (1) is preferably as follows: non-starch polysaccharide 10 mg/mL, yeast extract 2.0 mg/mL, peptone 2.0 mg/mL, bile salt 0.5 mg/mL, hemin chloride 0.02 mg/mL, L-cysteine 0.5 mg/mL, sodium bicarbonate 2.0 mg/mL, sodium chloride 0.1 mg/mL, calcium chloride hexahydrate 0.01 mg/mL, magnesium sulfate heptahydrate 0.01 mg/mL, dipotassium hydrogen phosphate 0.04 mg/mL, potassium dihydrogen phosphate 0.04 mg/mL, vitamin _K1 0.01 mg/mL, resazurin 0.01 mg/mL and Tween 80 2.0 mg/mL, pH value is 7.0-7.4, and water is used as solvent.

The pH value is preferably 7.2.

The sterilization conditions described in step (1) are preferably sterilized at 121° C. for 15 to 30 minutes.

The feces described in step (1) were obtained from three healthy adult volunteers who had no digestive diseases or dietary restrictions, had not taken any antibiotics in the past 3 months, and had not taken any probiotic products in the past 2 weeks.

The fecal flora solution described in step (1) is prepared by the following steps: the collected fecal sample is immediately placed in an anaerobic chamber, and the fecal sample is mixed evenly with saline in an amount of feces: saline = 1 g: 9 mL, and then filtered through four layers of gauze to obtain a fecal flora solution.

The inoculation amount of the fecal flora solution in step (1) is preferably 10% by volume.

The conditions of the anaerobic fermentation in step (1) are preferably constant temperature at 36-38° C. and constant pH for 7-9 hours; more preferably constant temperature at 37° C. and constant pH for 8 hours.

The solid-liquid separation method in step (1) is preferably centrifugation.

The centrifugation conditions are: centrifugation at 4°C and 12,000×g for 10 min.

The abundance of the bacterial biomarker described in step (2) is determined by real-time fluorescence quantitative PCR, preferably by the following steps:

A) extracting total DNA from microbial communities, testing the quality of DNA extraction using agarose gel electrophoresis, and determining the concentration and purity of DNA using an ultraviolet spectrophotometer;

B) determining the content of bacterial biomarkers in the fermentation precipitate obtained in step (1) by real-time fluorescence quantitative PCR;

The primers for real-time fluorescence quantitative PCR are:

Faecalibacterium upstream primer: 5′-GGAGGAAGAAGGTCTTCGG-3′;

Faecalibacterium downstream primer: 5′-AATTCCGCCTACCTCTGCACT-3′;

Coprococcus upstream primer: 5′-CCATGGAAGAGGTGGACCAT-3′;

Coprococcus downstream primer: 5′-TTTCTCATGATGCCCGCAAG-3′;

Bacteroides upstream primer: 5′-AAACCCATACGCCGCAAG-3′;

Bacteroides downstream primer: 5′-GACACCTCACGGCACGAG-3′;

Anaerostipes upstream primer: 5′-CCATTGTATGCGGCTCAGGA-3′;

Anaerostipes downstream primer: 5′-CAATAATCGGTGTCGGCCCT-3′;

338F: 5′-ACTCCTACGGGAGGCAGCAG-3′;

806R: 5’-GGACTACHVGGGTWTCTAAT-3’.

The system of real-time fluorescence quantitative PCR in step B) is as follows: 10 μL of 2×ChamQ Universal SYBR qPCR Master Mix, 0.4 μL of upstream primer (10 μM), 0.4 μL of downstream primer (10 μM), 10 ng of template DNA, and finally supplemented with ddH ₂ O to 20 μL.

The conditions of the real-time fluorescence quantitative PCR described in step B) are: pre-denaturation at 95°C for 30s; denaturation at 95°C for 10s, annealing at 60°C for 30s, and 40 cycles; the conditions for drawing the melting curve are: keeping at 95°C for 15s, keeping at 60°C for 60s, and keeping at 95°C for 15s.

The metabolite biomarker described in step (2) is preferably determined by liquid chromatography; more preferably, it is determined by the following steps: quantitatively using a standard substance, the specific steps are as follows:

① The liquid obtained in step (1) and the extract are mixed evenly, extracted by ultrasonic method at 4-5°C and 35-45KHz for 25-35min, and then allowed to stand at 18-25°C for 20-40min, and then centrifuged, the supernatant is removed, dried with nitrogen, and then re-dissolved with a re-solution, extracted by ultrasonic method at 4-5°C and 35-45KHz for 3-6min, centrifuged, and the supernatant is collected into an injection vial; the extract is obtained by mixing acetonitrile and methanol in a volume ratio of 1:1; the re-solution is obtained by mixing acetonitrile and water in a volume ratio of 1:1;

②Chromatographic column: BEH C18 chromatographic column, injection volume: 10 μL, mobile phase A is a 0.1% by volume formic acid aqueous solution, mobile phase B is an acetonitrile/isopropanol solution mixed in a volume ratio of 1:1, containing 0.1% by volume formic acid; flow rate is 0.40 mL/min, column temperature is 40°C;

③ Separation gradient: 0-3min, mobile phase A decreases from linear 95% to 80%, and mobile phase B increases from linear 5% to 20%; 3-9min, mobile phase A decreases from linear 80% to 5%, and mobile phase B increases from linear 20% to 95%; 9-13min, mobile phase A maintains linearity at 5%, and mobile phase B maintains linearity at 95%; 13.01-3.1min, mobile phase A increases linearity from 5% to 95%, and mobile phase B decreases linearity from 95% to 5%; 13.1-16min, mobile phase A maintains linearity at 95%, and mobile phase B maintains linearity at 5%.

The amount of the extract in step ① is preferably in a ratio of liquid:extract = 1:3-5 by volume; more preferably in a ratio of liquid:extract = 1:4 by volume.

The conditions of ultrasonic extraction described in step ① are preferably 5° C., 40 KHz ultrasonic extraction for 30 min.

The standing condition described in step ① is preferably 20° C., 30 min.

The centrifugation conditions described in step ① are preferably at 4° C., 13,000×g for 15 min.

The amount of the reconstitution solution in step ① is preferably 120 μL.

The specification of the BEH C18 chromatographic column described in step ② is 100 mm×2.1 mm, and the pore size is 1.8 μm.

Use of the above biomarkers or the above methods in the preparation of hypoglycemic preparations.

Compared with the prior art, the present invention has the following advantages and effects:

The present invention provides a group of biomarkers, including Faecalibacterium, Coprococcus, Bacteroides, Anaerostipes, ginger sugar lipid A, 1-phosphoterin, matrine, tricarballylic acid, N-carbamyl putrescine, Nω-acetylhistamine, tyramine, and benzamide, which can be used to screen non-starch polysaccharides with strong hypoglycemic activity, and the steps are simple and easy to implement, which is conducive to reducing experimental and detection costs;

Under the in vitro anaerobic fermentation conditions described in the present application, the abundance of metabolite biomarkers obtained after fermentation of non-starch polysaccharides can be used to quickly determine the hypoglycemic activity of unknown non-starch polysaccharides, which is conducive to saving time in discovering and screening non-starch polysaccharides with hypoglycemic activity, and promoting the rapid development and application of medicines, food, feed and other products based on non-starch polysaccharides.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an S-chart of different metabolites in different samples and control groups under anion or cation mode based on multidimensional statistical analysis of the OPLS-DA model; wherein the ordinate represents the correlation coefficient between the principal component and the metabolite, the abscissa represents the co-correlation coefficient between the principal component and the metabolite, the red dots represent that the VIP values of these metabolites are greater than or equal to 1, and the green dots represent that the VIP values of the metabolites are less than 1.

Figure 2 is a volcano plot of differential metabolites of Ganoderma lucidum polysaccharide, Lentinan, Cordyceps militaris polysaccharide and the control group; wherein the horizontal axis is the fold change value of the metabolite expression difference between the two groups, i.e., log ₂ FC, and the vertical axis is the statistical test value of the difference in metabolite expression, i.e., -log ₁₀ (p value).

Figure 3 is a heat map of the relative abundance of bacterial flora at the genus level (top 30) in Ganoderma lucidum polysaccharide, Lentinan polysaccharide, Cordyceps militaris polysaccharide and the control group.

Figure 4 is a heat map showing the correlation between the top 30 bacteria in relative abundance and the 14 potential key metabolites.

Detailed ways

The present invention will be described in further detail below in conjunction with examples and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out under conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially. The chemical reagents involved in the present invention are all analytically pure.

Ganoderma lucidum, Cordyceps militaris, Lentinus edodes, and Poria cocos were purchased from Beijing Tong Ren Tang Co., Ltd.; neutral protease was purchased from Shanghai Yuanye Biotechnology Co., Ltd., CAS No.: 9068-59-1; human liver cancer cells (HepG2 cells) and insulin were purchased from Wuhan Pronocell Life Science Co., Ltd.; metformin was purchased from Shanghai Suyuan Biotechnology Co., Ltd.

The feces were obtained from three healthy adult volunteers who had no digestive diseases or dietary restrictions, had not taken any antibiotics in the past 3 months, and had not taken any probiotic products in the past 2 weeks. In each example, the feces samples from these three healthy adult volunteers were used for inoculation, and the results obtained were substantially the same.

Example 1

(1) In vitro fermentation of edible fungus polysaccharides:

The polysaccharide in Ganoderma lucidum is extracted by ethanol defatting, hot water extraction, neutral protease treatment, sevag method protein removal, ethanol precipitation, dialysis, and freeze drying. The specific steps are as follows

1) Soak the Ganoderma lucidum in 80% (v/v) ethanol solution for 24 h (solid-liquid ratio is 1 g: 4 mL) to defat it, and then place the residue in a 60°C oven for 1 h after filtration;

2) Mix the defatted and dried Ganoderma lucidum with ultrapure water at a ratio of 1 g of raw material to 20 mL of ultrapure water, and extract twice at 100° C. for 2 hours each time. After the first extraction, filter the aqueous solution with four layers of gauze, and extract the obtained filter residue with water under the same conditions as the first extraction, and combine the two filtrates;

3) adding neutral protease (final concentration 0.5% w/v) to the filtrate and reacting at 50°C for 4 h;

4) Sevag method (chloroform and n-butanol were mixed in a volume ratio of 4:1 to obtain a chloroform/n-butanol mixture, and the polysaccharide solution and the chloroform/n-butanol mixture were mixed in a volume ratio of 1:4, shaken for 30 minutes each time, and centrifuged at 4000 rpm for 1 minute) to remove protein three times, and the supernatant was concentrated by rotary evaporator to remove residual chloroform and n-butanol;

5) Add anhydrous ethanol in a ratio of concentrate to anhydrous ethanol = 1:4 by volume, and precipitate at 4°C overnight. Collect the precipitate, and add water (the amount added is equal to the volume of the original concentrate) to redissolve the precipitate to obtain a polysaccharide solution;

6) The supernatant was dialyzed (the molecular weight cut-off of the dialysis bag was 10000Da) and vacuum freeze-dried to obtain Ganoderma lucidum polysaccharide.

Ganoderma lucidum polysaccharide and water were mixed at a ratio of 1 g of Ganoderma lucidum polysaccharide: 100 mL of water, and yeast extract (final concentration of 2.0 mg/mL), peptone (final concentration of 2.0 mg/mL), bile salts (final concentration of 0.5 mg/mL), hemin chloride (final concentration of 0.02 mg/mL), L-cysteine (final concentration of 0.5 mg/mL), sodium bicarbonate (final concentration of 2.0 mg/mL), sodium chloride (final concentration of 0.1 mg/mL), calcium chloride hexahydrate (final concentration of 0.01 mg/mL), magnesium sulfate heptahydrate (final concentration of 0.01 mg/mL), dipotassium hydrogen phosphate (final concentration of 0.04 mg/mL), potassium dihydrogen phosphate (final concentration of 0.04 mg/mL), vitamin K ₁ (final concentration of 0.01 mg/mL), resazurin (final concentration of 0.01 mg/mL) and Tween 80 (final concentration of 2.0 mg/mL), mix evenly, adjust the pH to 7.2, and sterilize at high temperature and high pressure (sterilize at 121°C for 30 min) to obtain a fermentation medium. Use physiological saline to mix the fecal sample evenly (feces/physiological saline 1:9 w/v), and then filter through four layers of gauze to obtain a fecal flora solution. The fecal flora solution was inoculated into the fermentation medium with an inoculation volume of 10%, and anaerobically, constantly, and at a constant pH for 8 hours at 37°C (using a fully automatic fermentation control system (Beijing Mancang Technology Co., Ltd.), using 2 mol/L sulfuric acid and 2 mol/L sodium hydroxide configured in advance, to achieve constant pH adjustment, and to ensure an anaerobic environment by continuously passing filtered and sterilized _N2 ), centrifuged at 4°C and 12000×g for 10 minutes to obtain a fermentation precipitate and a fermentation supernatant of Ganoderma lucidum polysaccharide, i.e., the sample of Example 1. The control samples were processed in the same manner as the samples except that no additional carbon source (ie, Ganoderma lucidum polysaccharide) was added to the control samples, and they were fermented and centrifuged under the same conditions.

(2) Biomarker determination:

1) Extract total DNA from microbial communities, detect the extraction quality of DNA using agarose gel electrophoresis, and determine the concentration and purity of DNA using Nanodrop.

2) determining the content of bacterial biomarkers in the fermentation precipitate obtained in step (1) by real-time fluorescence quantitative PCR;

The primers for real-time fluorescence quantitative PCR are:

Faecalibacterium upstream primer: 5′-GGAGGAAGAAGGTCTTCGG-3′;

Faecalibacterium downstream primer: 5′-AATTCCGCCTACCTCTGCACT-3′;

Coprococcus upstream primer: 5′-CCATGGAAGAGGTGGACCAT-3′;

Coprococcus downstream primer: 5′-TTTCTCATGATGCCCGCAAG-3′;

Bacteroides upstream primer: 5′-AAACCCATACGCCGCAAG-3′;

Bacteroides downstream primer: 5′-GACACCTCACGGCACGAG-3′;

Anaerostipes upstream primer: 5′-CCATTGTATGCGGCTCAGGA-3′;

Anaerostipes downstream primer: 5′-CAATAATCGGTGTCGGCCCT-3′;

Agathobacter upstream primer: 5′-GCTAAATACGTGCCAGCAGC-3′;

Agathobacter downstream primer: 5′-AATGCAGTACCGGGGTTGAG-3′;

338F: 5′-ACTCCTACGGGAGGCAGCAG-3′;

806R: 5’-GGACTACHVGGGTWTCTAAT-3’.

Real-time fluorescence quantitative PCR was performed using the ChamQ Universal SYBR qPCR MasterMix kit from Nanjing Novozymes Biotech Co., Ltd. The system of real-time fluorescence quantitative PCR was as follows: 2×ChamQ UniversalSYBR qPCR Master Mix 10 μL, upstream primer (10 μM) 0.4 μL, downstream primer (10 μM) 0.4 μL, template DNA 10 ng, and finally supplemented to 20 μL with ddH ₂ O.

The conditions for real-time fluorescence quantitative PCR were as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and 40 cycles; the conditions for drawing the melting curve were as follows: 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s.

3) Determine the content of the remaining metabolite biomarkers in the fermentation supernatant in step (1) by liquid chromatography, and quantify them using standard products, wherein the standard products are ginger glycolipid A, 1-phosphoterin, matrine, tricarbamic acid, N-carbamoyl putrescine, Nω-acetylhistamine, tyramine, and benzamide, and the specific steps are as follows:

A. Take 100 μL of fermentation supernatant in a 1.5 mL centrifuge tube, add 400 μL of extracting solution (acetonitrile: methanol = volume ratio 1:1), vortex mix, extract by low-temperature ultrasound (5°C, 40 KHz) for 30 min, let stand at 20°C for 30 min, then centrifuge at 4°C, 13000 × g for 15 min, remove the supernatant, blow dry with nitrogen, and then re-dissolve with 120 μL of re-solution (acetonitrile: water = volume ratio 1:1), extract by low-temperature ultrasound for 5 min (5°C, 40 KHz), centrifuge at 4°C, 13000 × g for 5 min, and collect the supernatant into a sampling vial;

B. Chromatographic column: BEH C18 column (100 mm × 2.1 mm, 1.8 μm), injection volume: 10 μL, mobile phase A: water (containing 0.1% v/v formic acid), mobile phase B: acetonitrile/isopropanol (volume ratio 1:1, containing 0.1% v/v formic acid), flow rate: 0.40 mL/min, column temperature: 40 °C;

C. Separation gradient: 0-3min, mobile phase A decreases from 95% to 80% linearly, mobile phase B increases from 5% to 20% linearly; 3-9min, mobile phase A decreases from 80% to 5% linearly, mobile phase B increases from 20% to 95% linearly; 9-13min, mobile phase A maintains linearity at 5%, mobile phase B maintains linearity at 95%; 13.0-13.1min, mobile phase A increases linearity from 5% to 95%, mobile phase B decreases linearity from 95% to 5%; 13.1-16min, mobile phase A maintains linearity at 95%, mobile phase B maintains linearity at 5%.

(4) Screening and evaluation: The hypoglycemic activity of edible fungi polysaccharides from different sources was screened and evaluated based on the abundance of bacterial biomarkers and the differential metabolism of multiple metabolite biomarkers.

The results are shown in Table 1. The total abundance T1 of bacterial biomarkers in the sample of Example 1 was 32.00±0.16%, and the total concentration T2 of ginger lipid A, 1-phosphoterin, matrine, tricarbamic acid, N-carbamoyl putrescine, Nω-acetylhistamine, tyramine, and benzamide was 42.63±1.08 mM, satisfying: T1≥18.0*TC1 and T2≥1.18*TC2;, therefore, the sample of Example 1 had a stronger hypoglycemic effect, that is, the Ganoderma lucidum polysaccharide had a stronger hypoglycemic effect.

Table 1 Biomarker levels in the Ganoderma lucidum polysaccharide fermentation broth prepared in Example 1

(5) Evaluation of hypoglycemic effect: The sample of Example 1 and the control sample were filtered through a 0.22 μm filter membrane and then diluted with DMEM complete medium (DMEM medium containing 10% v/v fetal bovine serum) to form a 0.625% (v/v) solution as the test sample. Their hypoglycemic ability in human liver cancer cells was compared. The specific steps were as follows:

Take the logarithmic phase HepG2 cells in good condition, blow them down and transfer them to a 15mL centrifuge tube, centrifuge at 800rpm for 4min, remove the supernatant, add new DMEM complete culture medium to resuspend the cells, and gently blow the cells to disperse them evenly. Use Countess automatic cell counter to count the cells, blow the cells thoroughly, mix them, pipette 10μL of cell solution into the centrifuge tube, add an equal amount of 0.4% trypan blue staining solution, blow them thoroughly, pipette 10μL into the Countess cell counting plate, let it stand for 30 seconds, insert the cell counting plate into the Countess cell counter, and record the number of live cells. The automatic cell counter requires that the cell density should not be too large during measurement. If necessary, the cells can be diluted or more culture medium can be added to the appropriate density.

According to the results of cell density determination, the cell concentration was appropriately diluted to adjust to 1×10 ⁶ cells/mL, and the cells were seeded in a 96-well plate at a density of 2×10 ⁴ cells per well. After 12 hours of culture, the culture medium was replaced with DMEM culture medium containing fermentation supernatant dissolved at different concentrations (0, 12.5, 25, 25, 50, 100, 200 μg/L). After 24 hours of culture, the culture medium was replaced with 100 μL of 10% CCK-8 solution in each well. After 2 hours, the absorbance of each well was measured at a wavelength of 450 nm using an ELISA reader, and the cell viability of each group was calculated. The optimal concentration that was non-cytotoxic to HepG2 cells was selected for subsequent experiments.

HepG2 cells in the logarithmic growth phase were seeded in 96-well plates at a rate of 2×10 ⁴ cells per well and cultured for 12 hours. Then, the cells were washed with phosphate buffer (0.01 mol/L, pH 7.4), and the culture medium was replaced with DMEM medium containing different concentrations of insulin (0, 0.2, 1, 5, 25, 125 μg/L). After the cells were treated for 24 hours, 36 hours, and 48 hours, the glucose concentration in the culture medium was detected using a glucose oxidase assay kit. The glucose consumption (mg) was calculated by the difference in glucose concentration at the beginning and end of the experiment. The optimal conditions for establishing the insulin-resistant HepG2 model were determined based on glucose consumption.

After establishing the insulin-resistant HepG2 model according to the assay conditions, the cells were washed three times with DMEM medium without fetal bovine serum. 100 μL of DMEM medium containing the optimal concentration of fermentation supernatant was added to treat the cells. Normal HepG2 cells were used as blank cells and were referred to as the normal group. The negative control was insulin-resistant HepG2 cells, referred to as the model group, and the group treated with fermentation supernatant was the experimental group. After 24 hours of culture, the supernatant of the culture medium was collected and the glucose concentration was detected using a glucose detection kit. In addition, the glucose consumption of HepG2 cells in different groups was calculated by the difference in glucose concentration at the beginning and end of the experiment.

The protein concentration of each group of cell homogenate was determined using a protein concentration detection kit. According to the instructions of the cell glycogen, hexokinase and phosphoenolpyruvate carboxykinase detection kits, the glycogen content and the enzyme content closely related to sugar metabolism in each group were determined.

All the above experiments were repeated three times or more. One-way ANOVA was performed using GraphPad Prism 9.0.0 software, and the data of each group were expressed as mean ± standard deviation (x ± s). Duncan's method was used to compare the differences between groups, and the significance level p < 0.05 was considered significant.

The results are as follows: The glucose consumption of insulin-resistant HepG2 cells treated with the sample of Example 1 was significantly different from that of the control group. The glucose consumption increased by 34.44%, the glycogen content increased by 35.34%, the hexokinase increased by 7.33%, and the phosphoenolpyruvate carboxykinase decreased by 12.72%. The above indicates that Ganoderma lucidum polysaccharide has a strong hypoglycemic activity.

Example 2 is basically the same as Example 1, except that the edible fungus polysaccharide is replaced by Cordyceps militaris polysaccharide.

(1) In vitro fermentation of edible fungi polysaccharides: water was added at a ratio of 1 g of Cordyceps militaris polysaccharide: 100 mL of water, and yeast extract, peptone, bile salts, hemin chloride, L-cysteine, sodium bicarbonate, sodium chloride, calcium chloride hexahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium dihydrogen phosphate, vitamin _K1 , resazurin and Tween 80 were added at the required concentrations, mixed evenly, pH adjusted, and sterilized at high temperature and high pressure to obtain a fermentation medium. A fecal sample was homogenized with physiological saline (feces/physiological saline 1:9 w/v), and then filtered through four layers of gauze to obtain a fecal flora solution. The fecal flora solution was inoculated into the fermentation medium with an inoculation volume of 10%, and anaerobically, constantly, and at a constant pH for 8 h at 37 ° C was fermented, and the fermentation precipitate and fermentation supernatant of Cordyceps militaris polysaccharide were obtained by centrifugation, i.e., the sample of Example 2. The treatment method of the control sample was basically the same as that of the sample, except that the control sample did not add an additional carbon source, and was fermented and centrifuged under the same conditions.

(2) Biomarker determination: Determine the content of bacterial biomarkers in the fermentation precipitate of step (1) by real-time fluorescence quantitative PCR; Determine the content of metabolite biomarkers in the fermentation supernatant of step (1) by liquid chromatography.

(3) Screening and evaluation: The hypoglycemic activity of edible fungi polysaccharides from different sources was screened and evaluated based on the abundance of bacterial biomarkers and the differential metabolism of multiple metabolite biomarkers.

The results are shown in Table 2. The total abundance T1 of bacterial biomarkers in the sample of Example 2 was 3.93±0.01%, and the total concentration T2 of ginger lipid A, 1-phosphoterin, matrine, tricarbamic acid, N-carbamoyl putrescine, Nω-acetylhistamine, tyramine, and benzamide was 39.84±0.50 mM, which satisfied: 2.40*TC1≤T1＜3.2*TC1 and 1.06*TC2≤T2＜1.14*TC2, so the hypoglycemic effect of the sample of Example 2 was weak, that is, the hypoglycemic effect of Cordyceps militaris polysaccharide was weak.

Table 2 Biomarker levels in the Cordyceps militaris polysaccharide fermentation broth prepared in Example 2

(4) Evaluation of hypoglycemic effect: The operation steps were the same as those in Example 1. The results were as follows: The glucose consumption of insulin-resistant HepG2 cells treated with the samples of this example was different from that of the control group. The glucose consumption increased by 17.18%, the glycogen content increased by 10.64%, and the phosphoenolpyruvate carboxykinase decreased by 2.70%. The above indicated that the hypoglycemic activity of Cordyceps militaris polysaccharide was weak.

Example 3 is basically the same as Example 1, except that the source of edible fungus polysaccharide is replaced by shiitake mushrooms.

(1) In vitro fermentation of edible fungus polysaccharides: water was added at a ratio of 1 g of lentinan to 100 mL of water, and yeast extract, peptone, bile salt, hemin chloride, L-cysteine, sodium bicarbonate, sodium chloride, calcium chloride hexahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium dihydrogen phosphate, vitamin _K1 , resazurin and Tween 80 were added at the required concentrations, mixed evenly, pH adjusted, and sterilized at high temperature and high pressure to obtain a fermentation medium. The fecal sample was homogenized with physiological saline (feces/physiological saline 1:9 w/v), and then filtered through four layers of gauze to obtain a fecal flora solution. The fecal flora solution was inoculated into the fermentation medium with an inoculation volume of 10%, and anaerobically, constantly, and at a constant pH at 37°C for 8 hours. The fermentation precipitate and fermentation supernatant of lentinan were obtained by centrifugation, i.e., the sample of Example 3. The treatment of the control sample was basically the same as that of the sample, except that no additional carbon source was added to the control sample, and it was fermented and centrifuged under the same conditions.

(2) Biomarker determination: Biomarker determination: Determine the content of bacterial biomarkers in the fermentation precipitate of step (1) by real-time fluorescence quantitative PCR; Determine the content of metabolite biomarkers in the fermentation supernatant of step (1) by liquid chromatography.

(3) Screening and evaluation: The hypoglycemic activity of edible fungi polysaccharides from different sources was screened and evaluated based on the abundance of bacterial biomarkers and the differential metabolism of multiple metabolite biomarkers.

The results are shown in Table 3. The total abundance T1 of bacterial biomarkers in the sample of Example 3 was 27.51±0.06%, and the total concentration T2 of ginger lipid A, 1-phosphoterin, matrine, tricarbamic acid, N-carbamoyl putrescine, Nω-acetylhistamine, tyramine, and benzamide was 40.82±0.56 mM, which satisfied: 3.2*TC1≤T1＜18.0*TC1 and 1.14*TC2≤T2＜1.18*TC2, so the hypoglycemic effect of the sample of Example 3 was moderate, that is, the hypoglycemic effect of Lentinan was moderate.

Table 3 Biomarker levels in Lentinan fermentation broth prepared in Example 3

(4) Evaluation of hypoglycemic effect: The operation steps are the same as Example 1, and the results are as follows: The glucose consumption of insulin-resistant HepG2 cells treated with the samples of this example is different from that of the control group. The glucose consumption increases by 24.44%, the glycogen content increases by 19.83%, the hexokinase increases by 5.89%, and the phosphoenolpyruvate carboxykinase decreases by 2.30%. The above indicates that the hypoglycemic activity of Lentinan is moderate.

The effect embodiment is basically the same as embodiment 1, except that the source of edible fungus polysaccharide is replaced by Poria cocos

(1) In vitro fermentation of edible fungus polysaccharides:

The alkali-soluble polysaccharides in Poria cocos were extracted by ethanol defatting, hot water extraction, alkali solution treatment, supernatant collection, hydrochloric acid neutralization, dialysis, and freeze drying. The specific steps are as follows

1) Soak Poria cocos in 80% (v/v) ethanol solution for 24 h (solid-liquid ratio is 1 g: 4 mL) to defat, and then filter the residue and place it in a 60°C oven for 1 h;

2) Mix the defatted and dried Poria cocos with ultrapure water at a ratio of 1 g of raw material to 20 mL of ultrapure water, and extract twice at 100°C for 2 h each time, and finally collect the precipitate;

3) Dissolve the precipitate: extract with 1 mol/L NaOH = 1 g: 60 mL for 1 h and collect the supernatant;

4) neutralizing the supernatant of step (3) with 1 mol/L hydrochloric acid to neutrality;

5) dialyzing for 48 h using a dialysis bag with a cutoff of 10,000 Da, and vacuum freeze-drying to obtain alkali-soluble Poria cocos polysaccharide.

Water was added at a ratio of 1 g alkali-soluble Poria cocos polysaccharide: 100 mL of water, and yeast extract, peptone, bile salts, hemin chloride, L-cysteine, sodium bicarbonate, sodium chloride, calcium chloride hexahydrate, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, potassium dihydrogen phosphate, vitamin _K1 , resazurin and Tween 80 were added at the desired concentration, mixed evenly, pH was adjusted, and the fermentation medium was obtained after high temperature and high pressure sterilization. The fecal sample was uniformly mixed with physiological saline (feces/physiological saline 1:9w/v), and then filtered through four layers of gauze to obtain a fecal flora solution. The fecal flora solution was inoculated into the fermentation medium with an inoculation volume of 10%, and anaerobically, constantly and at a constant pH for 8 h at 37 ° C was fermented, and the fermentation precipitation and fermentation supernatant of the alkali-soluble Poria cocos polysaccharide were obtained by centrifugation, i.e., the sample of Example 4. The treatment of the control sample was basically the same as that of the sample, except that the control sample did not add an additional carbon source, and was fermented and centrifuged under the same conditions.

(2) Biomarker determination: Determine the content of bacterial biomarkers in the fermentation precipitate of step (1) by real-time fluorescence quantitative PCR; Determine the content of metabolite biomarkers in the fermentation supernatant of step (1) by liquid chromatography

(3) Screening and evaluation: The hypoglycemic activity of edible fungi polysaccharides from different sources was screened and evaluated based on the abundance of bacterial biomarkers and the differential metabolism of multiple metabolite biomarkers.

The results are shown in Table 4. The total abundance T1 of bacterial biomarkers in the sample of Example 4 was 4.94±0.51%, and the total concentration T2 of ginger lipid A, 1-phosphoterin, matrine, tricarbamic acid, N-carbamoyl putrescine, Nω-acetylhistamine, tyramine, and benzamide was 38.31±0.93 mM, which satisfied: 2.40*TC1≤T1＜3.2*TC1 and 1.06*TC2≤T2＜1.14*TC2, so the hypoglycemic effect of the sample of Example 4 was weak, that is, the hypoglycemic effect of alkali-soluble Poria cocos polysaccharide was weak.

Table 4 Biomarker levels in alkaline-soluble tuckahoe polysaccharide fermentation broth

(4) Evaluation of hypoglycemic effect: The operation steps were the same as Example 1, and the results were as follows: the glucose consumption of insulin-resistant HepG2 cells treated with the samples of this example was significantly different from that of the control group, with glucose consumption increasing by 16.85% and glycogen content increasing by 18.93%. The above indicated that the hypoglycemic activity of alkali-soluble Poria cocos polysaccharide was weak.

Example 4 Biomarker Screening Method:

(1) The relative abundance of bacteria during fermentation was determined by 16S rDNA sequencing analysis, which was performed as follows:

according to The total DNA of the microbial community was extracted according to the instructions of soil DNA kit (Omega Bio-tek, Norcross, GA, US). The extraction quality of DNA was detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined by NanoDrop2000. 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') were used to amplify the variable region of 16S rRNA gene V3-V4 by PCR. The amplification procedure was as follows: 95℃ pre-denaturation for 3min, 27 cycles (95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 45s), then 72℃ stable extension for 10min, and finally stored at 10℃ (PCR instrument: ABI /> 9700 model). The PCR reaction system was: 4 μL of 5×TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 μM), 0.8 μL of downstream primer (5 μM), 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and ddH ₂ O to 20 μL. Each sample was repeated 3 times.

The PCR products of the same sample were mixed and recovered using 2% agarose gel. The recovered products were purified using AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), detected by 2% agarose gel electrophoresis, and quantified using Quantus ^TM Fluorometer (Promega, USA). The library was constructed using NEXTflex ^TM Rapid DNA-Seq Kit (Bioo Scientific, USA): (1) adapter ligation; (2) using magnetic beads to screen and remove adapter self-ligated fragments; (3) using PCR amplification to enrich the library template; (4) using magnetic beads to recover the PCR products to obtain the final library. Sequencing was performed using Illumina's Miseq PE300/NovaSeq PE250 platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.).

The original sequencing sequence was quality controlled using fastp (https://github.com/OpenGene/fastp, version 0.20.0) software, and spliced using FLASH (http://www.cbcb.umd.edu/software/flash, version 1.2.7) software: bases with a quality value of less than 20 at the end of the reads were filtered, a 50 bp window was set, and if the average quality value in the window was less than 20, the back-end bases were cut off from the window, reads below 50 bp after quality control were filtered, and reads containing N bases were removed; based on the overlap relationship between PE reads, paired reads were spliced (merged) into a sequence with a minimum overlap length of 10 bp; the maximum mismatch ratio allowed in the overlap region of the spliced sequence was 0.2, and non-compliant sequences were screened; samples were distinguished based on the barcode and primers at both ends of the sequence, and the sequence direction was adjusted, the number of mismatches allowed for the barcode was 0, and the maximum number of primer mismatches was 2.

The UPARSE software (http://drive5.com/uparse/, version 7.1) was used to cluster the sequences into OTUs and remove chimeras based on a similarity of 97%. The RDP classifier (http://rdp.cme.msu.edu/, version 2.2) was used to annotate each sequence by species classification and to align it with the Silva 16S rRNA database (version 138), with the alignment threshold set at 70%.

(2) The metabolite content in the fermentation supernatant was determined by non-targeted metabolomics, and the following steps were performed:

100 μL of fermentation supernatant was placed in a 1.5 mL centrifuge tube, and 400 μL of extraction solution (acetonitrile: methanol = volume ratio 1:1) was added and vortexed to mix. After low-temperature ultrasonic extraction (5°C, 40 kHz) for 30 min, it was allowed to stand at 20°C for 30 min, and then centrifuged at 4°C and 13,000×g for 15 min. The supernatant was removed, dried with nitrogen, and then re-dissolved with 120 μL of re-solution (acetonitrile: water = volume ratio 1:1), low-temperature ultrasonic extraction for 5 min (5°C, 40 kHz), and centrifuged at 4°C and 13,000×g for 5 min. The supernatant was collected into an injection vial; equal volumes of all sample metabolites were mixed to prepare quality control samples (QC). During the instrument analysis, a QC sample was inserted into every 8 samples to examine the repeatability of the entire analysis process.

LC-MS analysis was performed using AB SCIEX's ultra-high performance liquid chromatography tandem time-of-flight mass spectrometry UPLC-TripleTOF system. 10 μL of sample was separated on a BEH C18 column (100 mm × 2.1 mm, 1.8 μm) and then entered the mass spectrometry detection. Chromatographic column: BEH C18 column (100mm×2.1mm, 1.8μm), injection volume: 10μL, mobile phase A: water (containing 0.1% v/v formic acid), mobile phase B: acetonitrile/isopropanol (volume ratio 1:1, containing 0.1% v/v formic acid), flow rate: 0.40mL/min, column temperature: 40℃; separation gradient: 0-3min, mobile phase A decreased from linear 95% to 80%, mobile phase B increased from linear 5% to 20%; 3-9min, mobile phase A decreased from linear 80% to 5%, mobile phase B increased from linear 20% to 95%; 913min, mobile phase A maintained linearity at 5%, mobile phase B maintained linearity at 95%; 13.0-13.1min, mobile phase A increased linearity from 5% to 95%, mobile phase B decreased linearity from 95% to 5%; 13.1-16min, mobile phase A maintained linearity at 95%, mobile phase B maintained linearity at 5%. The sample mass spectrometry signal was collected in positive and negative ion scanning mode, with a mass scanning range of m/z: 50-1000. The ion spray voltage was 5000V for positive ionization, 4000V for negative ionization, 80V for declustering, 50psi for spray gas, 50psi for auxiliary heating gas, 30psi for air curtain gas, 500℃ for ion source heating, and 20-60V for cyclic collision energy.

After the computer was completed, the LC-MS raw data was imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for baseline filtering, peak identification, integration, retention time correction, and peak alignment. Finally, a data matrix of retention time, mass-to-charge ratio, and peak intensity was obtained. The data matrix used the 80% rule to remove missing values, that is, retaining variables with more than 80% non-zero values in at least one group of samples, and then filling in the vacant values (filling the vacant values with the minimum value in the original matrix). In order to reduce the errors caused by sample preparation and instrument instability, the response intensity of the sample mass spectrometry peak was normalized by the sum normalization method to obtain the normalized data matrix. At the same time, the variables with a relative standard deviation (RSD) of QC samples > 30% were deleted, and log10 logarithmic processing was performed to obtain the final data matrix for subsequent analysis. At the same time, the MS and MSMS mass spectrometry information was matched with the metabolic public database HMDB (http://www.hmdb.ca/) and Metlin (https://metlin.scripps.edu/) database to obtain metabolite information.

The preprocessed data were uploaded to the MajorBio cloud platform (https://cloud.majorbio.com) for data analysis. The R package ropls (Version 1.6.2) was used for principal component analysis (PCA) and orthogonal least partial squares discriminant analysis (OPLS-DA), and 7 rounds of interactive validation were used to evaluate the stability of the model. In addition, student’s t test and difference fold analysis were performed. The selection of significantly different metabolites was based on the variable weight value (VIP) obtained by the OPLS-DA model and the student’s t test p value. Metabolites with VIP>1 and p<0.05 were significantly different metabolites.

(3) Screening biomarkers:

The S-chart of the multidimensional statistical analysis OPLS-DA model was used to analyze the differences in metabolites between each edible fungus polysaccharide fermentation group and the control group. In the S-chart of the OPLS-DA model (Figure 1), variables with variable projection importance (VIP) greater than 1 were selected, and then the student's t test (Figure 2) was further used to screen the variables with a fold change (FC value) greater than 1.3 and significant differences (P < 0.05) as up-regulated differential metabolites. In the cationic and anionic modes, 1577, 1489, and 1618 differential metabolites were screened from Examples 1-3, respectively. In order to screen out metabolites with potential hypoglycemic effects, the differential metabolites that were significantly upregulated in the fungal polysaccharide group but not significantly upregulated in the control group were taken as the union, and 22 metabolites with potential hypoglycemic effects were obtained. The 22 metabolites were correlated with the hypoglycemic indicators, and the results are shown in Table 5.

Table 5 Correlation between differential metabolite levels in fermentation broth and glucose-lowering related indicators

Note: * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001.

As can be seen from Table 5, the correlation between nutrients bile acid, heptanoic acid, ginger sugar lipid A, 1-phosphoterin, fluorescein acid D2, L-dihydroorotic acid, oxymatrine, D-pipecolic acid, 4-imidazolinone-5-propionic acid, tricarbamic acid, N-carbamoyl putrescine, and benzamide and the glucose content and glycogen content of insulin-resistant HepG2 cells was high, all greater than 0.5. Ginger sugar lipid A, 1-phosphoterin, oxymatrine, tricarbamic acid, N-carbamoyl putrescine, Nω-acetylhistamine, tyramine, and benzamide were also differential metabolites with significantly increased contents after acidic Poria polysaccharide fermentation.

The genus levels in each edible fungus polysaccharide fermentation group and the control group are shown in Figure 3. The top 30 bacteria in terms of genus abundance were analyzed for correlation with nutrients such as bile acid, heptanoic acid, ginger lipid A, 1-phosphoterin, fluorescein acid D2, L-dihydroorotic acid, matrine, D-piperidinic acid, 4-imidazolidinone-5-propionic acid, tricarbamic acid, N-carbamoylputrescine, and benzamide. The results are shown in Figure 4.

As can be seen from Figure 4, Faecalibacterium, Coprococcus, Bacteroides, and Anaerostipes showed a strong positive correlation with some key metabolites.

Therefore, the present invention identifies Faecalibacterium, Coprococcus, Bacteroides, Anaerostipes, ginger sugar lipid A, 1-phosphotin, oxymatrine, tricarbamic acid, N-carbamylputrescine, Nω-acetylhistamine, tyramine, and benzamide as biomarkers for screening edible fungus polysaccharides with hypoglycemic activity.

In addition, the present invention optimizes the method for determining the biomarkers, that is, using real-time fluorescence quantitative PCR to determine the content of bacterial markers Faecalibacterium, Coprococcus, Bacteroides, and Anaerostipes in in vitro fermentation of edible fungi polysaccharides, and using liquid chromatography in combination with standard products to determine the concentrations of ginger glycolipid A, 1-phosphoterin, matrine, tricarbamic acid, N-carbamyl putrescine, Nω-acetylhistamine, tyramine, and benzamide, the steps are as follows:

1) determining the content of bacterial biomarkers in the fermentation precipitate of step (1) by real-time fluorescence quantitative PCR;

The primers for real-time fluorescence quantitative PCR are:

Faecalibacterium upstream primer: 5′-GGAGGAAGAAGGTCTTCGG-3′;

Faecalibacterium downstream primer: 5′-AATTCCGCCTACCTCTGCACT-3′;

Coprococcus upstream primer: 5′-CCATGGAAGAGGTGGACCAT-3′;

Coprococcus downstream primer: 5′-TTTCTCATGATGCCCGCAAG-3′;

Bacteroides upstream primer: 5′-AAACCCATACGCCGCAAG-3′;

Bacteroides downstream primer: 5′-GACACCTCACGGCACGAG-3′;

Anaerostipes upstream primer: 5′-CCATTGTATGCGGCTCAGGA-3′;

Anaerostipes downstream primer: 5′-CAATAATCGGTGTCGGCCCT-3′;

338F: 5′-ACTCCTACGGGAGGCAGCAG-3′;

806R: 5’-GGACTACHVGGGTWTCTAAT-3’.

Real-time fluorescence quantitative PCR was performed using the ChamQ Universal SYBR qPCR MasterMix kit from Nanjing Novozymes Biotech Co., Ltd. The system of real-time fluorescence quantitative PCR was as follows: 2×ChamQ UniversalSYBR qPCR Master Mix 10 μL, upstream primer (10 μM) 0.4 μL, downstream primer (10 μM) 0.4 μL, template DNA 10 ng, and finally supplemented to 20 μL with ddH ₂ O.

The conditions for real-time fluorescence quantitative PCR were as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and 40 cycles; the conditions for drawing the melting curve were as follows: 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s.

2) Determine the content of the remaining metabolite biomarkers in the fermentation supernatant in step (1) by liquid chromatography, and quantify them using standard products, wherein the standard products are ginger glycolipid A, 1-phosphoterin, matrine, tricarbamic acid, N-carbamoyl putrescine, Nω-acetylhistamine, tyramine, and benzamide, and the specific steps are as follows:

A. Take 100 μL of fermentation supernatant in a 1.5 mL centrifuge tube, add 400 μL of extraction solution (acetonitrile: methanol = volume ratio 1:1), vortex mix, extract with low temperature ultrasound (5°C, 40KHz) for 30 min, let stand at 20°C for 30 min, then centrifuge at 4°C, 13000×g for 15 min, remove the supernatant, blow dry with nitrogen, and then re-dissolve with 120 μL of re-solution (acetonitrile: water = volume ratio 1:1), extract with low temperature ultrasound for 5 min (5°C, 40KHz), centrifuge at 4°C, 13000×g for 5 min, and collect the supernatant into a sampling vial;

B. Chromatographic column: BEH C18 column (100 mm × 2.1 mm, 1.8 μm), injection volume: 10 μL, mobile phase A: water (containing 0.1% v/v formic acid), mobile phase B: acetonitrile/isopropanol (volume ratio 1:1, containing 0.1% v/v formic acid), flow rate: 0.40 mL/min, column temperature: 40 °C;

C. Separation gradient: 0-3min, mobile phase A decreases from 95% to 80% linearly, mobile phase B increases from 5% to 20% linearly; 3-9min, mobile phase A decreases from 80% to 5% linearly, mobile phase B increases from 20% to 95% linearly; 9-13min, mobile phase A maintains linearity at 5%, mobile phase B maintains linearity at 95%; 13.0-13.1min, mobile phase A increases linearity from 5% to 95%, mobile phase B decreases linearity from 95% to 5%; 13.1-16min, mobile phase A maintains linearity at 95%, mobile phase B maintains linearity at 5%.

Then the total abundance of bacterial biomarkers Faecalibacterium, Coprococcus, Bacteroides and Anaerostipes was recorded as T1, and the total concentrations of ginger glycolipid A, 1-phosphopterin, matrine, tricarbamic acid, N-carbamoylputrescine, Nω-acetylhistamine, tyramine, benzamic acid, N-carbamoylbutanediamine, Nω-acetylhistamine, tyramine and benzamide were recorded as TC1 and TC2, respectively.

Ganoderma lucidum polysaccharide samples had the strongest hypoglycemic effect, and the abundance of biomarkers met T1≥18.0*TC1 and T2≥1.18*TC2. Cordyceps militaris polysaccharide and alkali-soluble Poria cocos polysaccharide samples had weaker hypoglycemic effects, and the abundance of biomarkers met 2.40*TC1≤T1＜3.2*TC1 and 1.06*TC2≤T2＜1.14. Lentinan polysaccharide had a moderate hypoglycemic effect and met 3.2*TC1≤T1＜18.0*TC1 and 1.14*TC2≤T2＜1.18*TC2.

Table 6 Ratio of the abundance of biomarkers in the example samples to the abundance of biomarkers in the control samples

Therefore, when T1≥18.0*TC1 and T2≥1.18*TC2 of the sample to be tested, the sample has a strong blood sugar lowering effect; when 3.2*TC1≤T1＜18.0*TC1 and 1.14*TC2≤T2＜1.18*TC2 of the sample to be tested, the sample has a moderate blood sugar lowering effect; when 2.40*TC1≤T1＜3.2*TC1 and 1.06*TC2≤T2＜1.14*TC2 of the sample to be tested, the sample has a weak blood sugar lowering effect; when T1＜2.40*TC1 and T2＜1.06*TC2 of the sample to be tested, the sample does not have the effect of alleviating diabetes.

The above embodiments are preferred implementations of the present invention, but the present invention is not limited to the specific embodiments described above. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (10)

1. A set of biomarkers for screening non-starch polysaccharides with hypoglycemic activity, characterized in that: it includes bacterial biomarkers and metabolic biomarkers; wherein: Bacterial biomarkers include: Faecalibacterium, Coprococcus, Bacteroides, Anaerostipes; Metabolite biomarkers included: ginger glycolipid A, 1-phosphoterin, matrine, tricarbamic acid, N-carbamylputrescine, Nω-acetylhistamine, tyramine, and benzamide. 2. Use of the biomarker for screening non-starch polysaccharides with hypoglycemic activity according to claim 1 in screening non-starch polysaccharides with strong hypoglycemic activity. 3. A method for screening non-starch polysaccharides with high hypoglycemic activity, characterized by comprising the following steps: (1) In vitro fermentation of non-starch polysaccharides: using the non-starch polysaccharide to be tested as the sole carbon source to prepare a fermentation medium; sterilizing or disinfecting the obtained fermentation medium, inoculating a fecal flora solution and performing in vitro anaerobic fermentation; separating the obtained fermentation liquid into solid and liquid to obtain a solid and a liquid; and setting a control, which is a fermentation medium without non-starch polysaccharides; (2) Biomarker determination: using real-time fluorescence quantitative PCR to determine the abundance of bacterial biomarkers in the solid obtained in step (1); determining the concentration of metabolite biomarkers in the liquid obtained in step (1); wherein the total abundance of bacterial biomarkers is T1, and the total concentration of ginger lipid A, 1-phosphoterin, matrine, tricarbamic acid, N-carbamyl putrescine, Nω-acetylhistamine, tyramine, and benzamide is T2; (3) Screening and evaluation: The hypoglycemic activity of the non-starch polysaccharide to be tested is obtained based on the abundance of bacterial biomarkers and the content of metabolite biomarkers combined with the following judgment criteria; judgement standard: ① If the following conditions are met, the non-starch polysaccharide to be tested is judged to have a strong hypoglycemic effect: T1 ≥ 18.0*TC1 and T2 ≥ 1.18*TC2; ② If the following conditions are met, the hypoglycemic effect of the non-starch polysaccharide to be tested is judged to be moderate: 3.2*TC1≤T1＜18.0*TC1 and 1.14*TC2≤T2＜1.18*TC2; ③ If the following conditions are met, the non-starch polysaccharide to be tested has a weak hypoglycemic effect: 2.40*TC1≤T1＜3.2*TC1 and 1.06*TC2≤T2＜1.14*TC2; ④ If the following conditions are met, it is determined that the hypoglycemic effect of the non-starch polysaccharide to be tested is not significant: T1＜2.40*TC1 and T2＜1.06*TC2. 4. The method for screening non-starch polysaccharides with high hypoglycemic activity according to claim 1, characterized in that: The non-starch polysaccharide in step (1) is derived from at least one of Ganoderma lucidum, Cordyceps militaris, Poria cocos and Lentinula edodes; The composition of the fermentation medium in step (1) is as follows: non-starch polysaccharide 10 mg/mL, yeast extract 2.0 mg/mL, peptone 2.0 mg/mL, bile salt 0.5 mg/mL, hemin chloride 0.02 mg/mL, L-cysteine 0.5 mg/mL, sodium bicarbonate 2.0 mg/mL, sodium chloride 0.1 mg/mL, calcium chloride hexahydrate 0.01 mg/mL, magnesium sulfate heptahydrate 0.01 mg/mL, dipotassium hydrogen phosphate 0.04 mg/mL, potassium dihydrogen phosphate 0.04 mg/mL, vitamin _K1 0.01 mg/mL, resazurin 0.01 mg/mL and Tween 80 2.0 mg/mL, pH value is 7.0-7.4, and water is used as solvent; The feces described in step (1) were collected from three healthy adult volunteers, who had no digestive diseases or dietary restrictions, had not taken any antibiotics in the past 3 months, and had not taken any probiotic products in the past 2 weeks; The fecal flora solution described in step (1) is prepared by the following steps: the collected fecal sample is immediately placed in an anaerobic chamber, and the fecal sample is mixed evenly with physiological saline in an amount of feces: physiological saline = 1 g: 9 mL, and then filtered through four layers of gauze to obtain a fecal flora solution; The inoculation amount of the fecal flora solution in step (1) is 10% by volume; The conditions of the anaerobic fermentation in step (1) are constant temperature of 36-38° C. and constant pH for anaerobic fermentation for 7-9 hours; The abundance of the bacterial biomarker described in step (2) is determined by real-time fluorescence quantitative PCR; The metabolite biomarkers described in step (2) are preferably measured by liquid chromatography. 5. The method for screening non-starch polysaccharides with high hypoglycemic activity according to claim 3, characterized in that: When the non-starch polysaccharide described in step (1) is a water-soluble polysaccharide, it is extracted by the steps of ethanol defatting, hot water extraction, neutral protease treatment, Sevag method protein removal, ethanol precipitation, dialysis, and freeze-drying; When the non-starch polysaccharide described in step (1) is an alkali-soluble polysaccharide, it is extracted through the steps of ethanol defatting, hot water extraction, alkali solution extraction, acid neutralization, dialysis, and freeze-drying. 6. The method for screening non-starch polysaccharides with high hypoglycemic activity according to claim 5, characterized in that: When the non-starch polysaccharide described in step (1) is a water-soluble polysaccharide, it is prepared by the following steps: 1) soaking the edible fungi in an ethanol solution with a concentration of 70 to 90% (v/v) to defatted and dried them to obtain a defatted and dried raw material; 2) mixing the defatted and dried raw material with water in a ratio of 1 g of the defatted and dried raw material to 15 to 30 mL of water, and leaching at 80 to 100° C. to obtain an extract; 3) then adding neutral protease to the extract to carry out enzymatic hydrolysis; reacting at 50°C for 4 hours; 4) removing protein from the enzymatic hydrolyzate obtained in step (3) by the Sevag method, removing organic reagents by a vacuum rotary evaporator and concentrating; 5) precipitating the concentrated solution obtained in step 4) with anhydrous ethanol at 4° C. overnight, collecting the precipitate by centrifugation, and re-dissolving the precipitate by adding water; 6) dialyzing and vacuum freeze-drying the supernatant to obtain non-starch polysaccharides; When the non-starch polysaccharide described in step (1) is an alkali-soluble polysaccharide, it is prepared by the following steps: A. soaking the edible fungi in an ethanol solution with a concentration of 70 to 90% (v/v) to defatted and dried them to obtain a defatted and dried raw material; B. Mix the defatted and dried raw material with water in a ratio of 1 g of defatted and dried raw material to 15 to 30 mL of water, and extract at 80 to 100° C. to obtain a precipitate; C. Leaching the precipitate with an alkaline solution to obtain a supernatant; D. neutralizing the supernatant obtained in step C with an acid; E. The neutralized supernatant is dialyzed and vacuum freeze-dried to obtain non-starch polysaccharides. 7. The method for screening non-starch polysaccharides with high hypoglycemic activity according to claim 6, characterized in that: The amount of the ethanol solution in step 1) is calculated based on a solid-liquid ratio of 1 g: 3 to 5 mL; The concentration of the ethanol solution in step 1) is 80% (v/v); The soaking time in step 1) is more than 12 hours; The drying condition in step 1) is drying at 50-70° C. for 30-90 min; The ratio described in step 2) is 1 g: 20 mL; The water described in step 2) is deionized water or ultrapure water; The extraction temperature in step 2) is 100° C. The extraction time in step 2) is 2h; The number of extractions in step 2) is preferably at least 2 times; The amount of the neutral protease used in step 3) is 0.3-1% (w/w) according to the concentration of the neutral protease in the extract; The conditions of the enzymatic hydrolysis reaction in step 3) are 40-60° C. for 3-5 hours; The reagent in the Sevag method described in step 4) is a reagent obtained by mixing chloroform and n-butanol in a volume ratio of 4:1; The number of times of protein removal in the Sevag method described in step 4) is 3 times; The concentration method in step 4) is rotary evaporation; The volume of anhydrous ethanol in step 5) is 3 to 5 times the volume of the concentrated solution; The dialysis bag in the dialysis described in step 6) is a dialysis bag with a molecular weight cut-off of 10000Da; The dialysis time in step 6) is 24 to 72 hours; The amount of the ethanol solution described in step A is calculated based on a solid-liquid ratio of 1 g: 3-5 mL; The concentration of the ethanol solution described in step A is 80% (v/v); The soaking time in step A is more than 12 hours; The drying condition described in step A is drying at 50-70° C. for 30-90 min; The ratio described in step B is 1 g:20 mL; The water described in step B is deionized water or ultrapure water; The leaching temperature in step B is 100°C; The extraction time described in step B is 2h; The number of extractions in step B is at least 2 times; The alkaline solution described in step C is a NaOH solution; The amount of the alkaline solution described in step C is calculated based on 50-70 mL of alkaline solution per gram of precipitation; The acid described in step D is hydrochloric acid; The degree of neutralization described in step D is neutralization to neutral; The dialysis bag in the dialysis described in step E is a dialysis bag with a molecular weight cut-off of 10000Da; The dialysis time in step E is 24 to 72 hours. 8. The method for screening non-starch polysaccharides with high hypoglycemic activity according to claim 3, characterized in that: The abundance of the bacterial biomarkers is determined by the following steps: A) extracting total DNA from microbial communities, testing the quality of DNA extraction using agarose gel electrophoresis, and determining the concentration and purity of DNA using an ultraviolet spectrophotometer; B) determining the content of bacterial biomarkers in the fermentation precipitate obtained in step (1) by real-time fluorescence quantitative PCR; The primers for real-time fluorescence quantitative PCR are: Faecalibacterium upstream primer: 5′-GGAGGAAGAAGGTCTTCGG-3′; Faecalibacterium downstream primer: 5′-AATTCCGCCTACCTCTGCACT-3′; Coprococcus upstream primer: 5′-CCATGGAAGAGGTGGACCAT-3′; Coprococcus downstream primer: 5′-TTTCTCATGATGCCCGCAAG-3′; Bacteroides upstream primer: 5′-AAACCCATACGCCGCAAG-3′; Bacteroides downstream primer: 5′-GACACCTCACGGCACGAG-3′; Anaerostipes upstream primer: 5′-CCATTGTATGCGGCTCAGGA-3′; Anaerostipes downstream primer: 5′-CAATAATCGGTGTCGGCCCT-3′; 338F: 5′-ACTCCTACGGGAGGCAGCAG-3′; 806R: 5′-GGACTACHVGGGTWTCTAAT-3′; The metabolite biomarker described in step (2) is determined by the following steps: quantification using a standard, the specific steps are as follows: ① The liquid obtained in step (1) and the extract are mixed evenly, extracted by ultrasonic method at 4-5°C and 35-45KHz for 25-35min, and then allowed to stand at 18-25°C for 20-40min, and then centrifuged, the supernatant is removed, dried with nitrogen, and then re-dissolved with a re-solution, extracted by ultrasonic method at 4-5°C and 35-45KHz for 3-6min, centrifuged, and the supernatant is collected into an injection vial; the extract is obtained by mixing acetonitrile and methanol in a volume ratio of 1:1; the re-solution is obtained by mixing acetonitrile and water in a volume ratio of 1:1; ②Chromatographic column: BEH C18 chromatographic column, injection volume: 10 μL, mobile phase A is a 0.1% by volume formic acid aqueous solution, mobile phase B is an acetonitrile/isopropanol solution mixed in a volume ratio of 1:1, containing 0.1% by volume formic acid; flow rate is 0.40 mL/min, column temperature is 40°C; ③ Separation gradient: 0-3min, mobile phase A decreases from linear 95% to 80%, and mobile phase B increases from linear 5% to 20%; 3-9min, mobile phase A decreases from linear 80% to 5%, and mobile phase B increases from linear 20% to 95%; 9-13min, mobile phase A maintains linearity at 5%, and mobile phase B maintains linearity at 95%; 13.01-3.1min, mobile phase A increases linearity from 5% to 95%, and mobile phase B decreases linearity from 95% to 5%; 13.1-16min, mobile phase A maintains linearity at 95%, and mobile phase B maintains linearity at 5%. 9. The method for screening non-starch polysaccharides with high hypoglycemic activity according to claim 8, characterized in that: The system of real-time fluorescence quantitative PCR described in step B) is as follows: 2×ChamQ Universal SYBR qPCR Master Mix 10 μL, 10 μM upstream primer 0.4 μL, 10 μM downstream primer 0.4 μL, template DNA 10 ng, and finally supplemented to 20 μL with ddH ₂ O; The conditions of the real-time fluorescence quantitative PCR described in step B) are: pre-denaturation at 95°C for 30s; denaturation at 95°C for 10s, annealing at 60°C for 30s, and 40 cycles; the conditions for drawing the melting curve are: 95°C for 15s, 60°C for 60s, and 95°C for 15s; The amount of the extract described in step ① is liquid: extract = volume ratio of 1:3-5; The ultrasonic extraction conditions described in step ① are 5° C., 40 KHz ultrasonic extraction for 30 min; The standing condition described in step ① is 20° C., 30 min; The centrifugation conditions described in step ① are 13000×g at 4°C for 15 min; The amount of the reconstitution solution described in step ① is 120 μL; The specification of the BEH C18 chromatographic column described in step ② is 100 mm×2.1 mm, and the pore size is 1.8 μm. 10. Use of the biomarker according to claim 1 or the method according to any one of claims 3 to 9 in the preparation of a hypoglycemic preparation.